Peptides capable of binding to the GAP protein SH3 domain, nucleotide sequences coding therefor, and preparation and use thereof

ABSTRACT

Peptides capable of interacting with the GAP protein SH3 domain, nucleic acid sequences coding therefor, and pharmaceutical compositions containing same, are disclosed.

The present invention relates to novel peptide and nucleotide sequencesand to their pharmaceutical use. More particularly, the inventionrelates to peptides which are able to bind to the SH3 domain of the GAPprotein.

The products of the Ras genes, generally designated p21 proteins, play akey role in the control of cell division in all the eucaryotic organismswhich have been investigated. Certain specific modifications of theseproteins cause them to lose their normal control and lead them to becomeoncogenic. Thus, a large number of human tumours are associated with thepresence of modified Ras genes. In the same way, overexpression of thesep21 proteins can lead to deregulation of cell proliferation. Takenoverall, the p21 proteins are implicated in 30% of human cancers.

An understanding of the precise role of these p21 proteins thereforeconstitutes one of the main objectives of research in the sphere ofoncology.

The model which is currently available for explaining the function ofthe p21 proteins rests on analogies which they share with the Gtransduction proteins. In cells, there is an equilibrium between theactive p21 proteins, which are bound to GTP, and the inactive forms,which have bound GDP. In a quiescent cell, where the p21 proteins arenot required, most of these proteins are in the GDP form. When the cellis stimulated, the nucleotide exchange factor, GEF, becomes more activeand promotes removal of the GDP and its replacement by GTP. The proteinthen adopts an active conformation which enables it to recognize andstimulate its effector, the GAP protein, "GTPase-activating protein",which is in all probability associated with other proteins. Thep21-GTP-GAP complex probably interacts, in turn, with one or more otherproteins, thereby resulting in transmission of the signal which leads toa biological response by the cell. The association of p21-GTP with GAPsimultaneously triggers hydrolysis of the GTP and return of the p21 toits inactive form.

In the case of the oncogenic p21 proteins, the mutation which they carryprevents return to the inactive state. In this latter case, theequilibrium is displaced towards the active form of p21.

This complex equilibrium between the active and inactive forms of p21 iscontrolled at one and the same time by factors which are inherent to thebiochemical properties of the p21 proteins (relative affinity for GDPand GTP, rate of nucleotide exchange, etc.) and external factors whichmodulate their activity, such as, in particular, the GAP protein.

The GAP protein is a cytosolic protein which is present in alleucaryotic organisms and which possesses, therefore, the property ofstrongly accelerating hydrolysis of the GTP which is bound to the normalp21 protein (Trahey and McCormick 1987). It possesses two domains whichare responsible for separate functions. Its carboxyterminal end carriesthe catalytic activity which binds the p21 proteins and increases theirGTPase activity. At its other end, downstream of the amino terminalpart, there is a juxtaposition of domains SH2 and SH3, which are able toparticipate in interactions with other proteins.

Currently, two proteins are known which interact with the GAP protein.These proteins are designated p62 and p190, being respectively of 62 kDaand 190 kDa molecular weight. Since these two proteins areimmunoprecipitated by antibodies directed against different epitopes ofGAP, they evidently form a specific complex with GAP. It is known, inparticular, that the p62 protein interacts with the GAP protein in theSH2 region.

As far as the SH3 domain is concerned, in particular, its presence invarious proteins such as the Cγ phospholipases (PLC-γ), the p85 subunitof 3-phosphatidylinositol kinase and the grb-2 protein, all of which areimplicated in transduction of the Ras p21 signal, suggests that thisdomain is of particular importance for directing protein/proteininteractions and therefore essential to the function of thecorresponding protein and/or its location in the cell. In the particularcase of the GAP protein, this SH3 domain could, therefore, alsoparticipate in transduction of the Ras signal. It is obvious that anunderstanding of the precise role of this SH3 domain would beparticularly valuable at the therapeutic level.

The object of the present invention is precisely that of contributing toelucidation of the contribution of the SH3 domain to transduction of theRas signal.

Thus, the Applicant has demonstrated the existence of proteins which areable to attach to the GAP protein by binding directly to its SH3 domain.

More specifically, the present invention results from theidentification, isolation and characterization of proteins which areable to interact with the SH3 domain of the GAP protein. It also resultsfrom the structural characterization of these proteins by identifyingcorresponding peptide sequences.

More especially, the polypeptide of the invention, which polypeptide isable to interact with the SH3 domain of the GAP protein, comprises allor part of a peptide sequence selected from among the sequences SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ IDNo. 9, or of a derivative of these sequences.

Within the meaning of the present invention, the term derivative denotesany molecule which is obtained by genetic and/or chemical modificationof the polypeptide according to the invention and which retains thesought-after activity. Genetic and/or chemical modification isunderstood to mean any mutation, substitution, deletion, addition and/ormodification of one or more residues. Such derivatives can be generatedfor different purposes such as, in particular, that of increasing theaffinity of the peptide for its site of interaction, that of improvingthe levels at which it is produced, that of increasing its resistance toproteases, that of increasing its therapeutic efficacy or of reducingits side-effects, or that of conferring on it novel pharmacokineticand/or biological properties.

It can also denote fragments of the abovementioned sequences ofderivatives of these fragments. Such fragments can be generated indifferent ways. In particular, they can be synthesized chemically, basedon the sequence given in FIG. 1, using the peptide synthesizers known tothe person skilled in the art. They can also be synthesized geneticallyby expressing a nucleotide sequence encoding the sought-after peptide ina cell host. In this case, the nucleotide sequence can be preparedchemically using an oligonucleotide synthesizer, based on the peptidesequence given in the present application and on the genetic code. Thenucleotide sequence can also be prepared, using the sequence given inthe present application, by enzymic restriction, ligation, cloning etc.,in accordance with the techniques known to the person skilled in theart, or by screening DNA libraries with probes developed on the basis ofSEQ ID No. 1.

According to one embodiment of the invention, the claimed peptidecomprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and/or SEQ ID No. 4.

Preferably, the polypeptide according to the invention has a molecularweight of the order of 68 kDa.

According to one embodiment of the invention, the polypeptide is apolypeptide of human origin comprising all or part of SEQ ID No. 5 orSEQ ID No. 9 or of one of their derivatives such as previously defined.

The polypeptide is, more especially, a polypeptide comprising SEQ ID No.5.

More preferably, the polypeptide is a polypeptide represented by SEQ IDNo. 9 or one of its derivatives.

Unexpectedly, this protein does not possess homology with another p68protein which has already been identified as binding to the SH3 domainof Src. Its tyrosine motifs are not phosphorylated in growing cells orin cells which are in a state of mitosis.

Analysis of the protein SEQ. ID No. 9 reveals that the protein which isattached to the SR+H3 domain of the GAP protein, and which is designatedG3BP, belongs to the hnRNP (heterologous nuclear ribonucleoproteins)family. More specifically, G3BP is a protein of 466 amino acids, whichhas an apparent molecular weight of 68 kDa and which contains severaldomains which are characteristic for proteins which bind to RNA:

RNP2 and RNP1 domains (amino acids 342 to 347)

4 RGG box (amino acids 435 to 449)

an acidic auxiliary domain (amino acids 144 to 221).

The invention also extends to peptides which are able to antagonize theinteraction between G3BP and the SH3 domain of GAP. The activity ofthese peptides can be demonstrated in competition tests (cf. Example2-3) or in tests involving interference with signals transduced by theRas proteins.

The invention also relates to polyclonal or monoclonal antibodies orantibody fragments which are directed against a polypeptide as definedabove. Such antibodies can be generated by methods known to the personskilled in the art. In particular, these antibodies can be prepared byimmunizing an animal against a polypeptide whose sequence is selectedfrom among SEQ ID No. 1, SEQ ID No. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQID No. 5 and SEQ ID No. 9 or any fragment or derivative of thesesequences, removing blood and isolating the antibodies. These antibodiescan also be generated by preparing hybridomas in accordance with thetechniques known to the person skilled in the art.

The antibodies or antibody fragments of the invention can then beemployed to regulate the activation state of the product of the Rasgenes.

Moreover, these antibodies can also be employed to detect and/or assay apeptide according to the invention in biological samples and, therefore,to provide information on the activation state of the product of the Rasgenes.

The invention also extends to antagonists, namely any peptide which isable to block the interaction of a polypeptide according to theinvention with the SH3 domain of the GAP protein. Such peptides can bedemonstrated in competition tests (cf. Example 2-3) or tests in whichRas activity is inhibited.

The present invention therefore renders it possible to generatepolypeptides which are derived from the sequences identified above andalso antibodies which are directed against these polypeptides orcorresponding proteins which exhibit biological properties which are ofinterest with a view to pharmaceutical utilization.

The invention also provides non-peptide compounds, or compounds whichare not exclusively peptide in nature, which can be usedpharmaceutically. Thus, it is possible, on the basis of the activeprotein motifs described in the present application, to constructmolecules which inhibit the p21 protein-dependent signal pathway andwhich are not exclusively peptide in nature and are compatible withpharmaceutical utilization. In this respect, the invention relates tothe use of a polypeptide of the invention, such as described above, forpreparing pharmacologically active non-peptide molecules, orpharmacologically active molecules which are not exclusively peptide innature, by determining the structural elements of this polypeptide whichare important for its activity and reproducing these elements by meansof non-peptide structures or of structures which are not exclusivelypeptide in nature. The invention also relates to pharmaceuticalcompositions which comprise one or more molecules which have beenprepared in this way.

The present invention also relates to any nucleic acid sequence encodinga polypeptide which is able to bind to the SH3 domain of the GAPprotein. More preferably, it relates to a sequence comprising:

(a) all or part of SEQ ID No. 6, or SEQ ID No. 10 or one of theircomplementary strands,

(b) any sequence which hybridizes with sequence (a) and encodes apolypeptide according to the invention, and

(c) the sequences which are derived from sequences (a) and (b) onaccount of the degeneracy of the genetic code.

Preferably, it comprises sequence SEQ ID No. 6 and, more preferably, isrepresented by SEQ ID No. 10.

The different nucleotide sequences of the invention may or may not be ofartificial origin. They can be genomic, cDNA or RNA sequences, hybridsequences or synthetic or semi-synthetic sequences. These sequences canbe obtained, for example, by screening DNA libraries (cDNA library orgenomic DNA library) using probes which are designed on the basis ofsequences presented above. Such libraries can be prepared from cells ofdifferent origins using conventional molecular biological techniquesknown to the person skilled in the art. The nucleotide sequences of theinvention can also be prepared by chemical synthesis, in particular inaccordance with the phosphoramidite method, or else by mixed methodsincluding chemical or enzymic modification of sequences obtained byscreening libraries.

These nucleotide sequences according to the invention may be used in thepharmaceutical domain, either for producing the polypeptides of theinvention, or for constructing antisense sequences which can be used ingene therapy, or else for detecting and diagnosing, by means ofhybridization experiments, the activity of the GAP protein in biologicalsamples, or for isolating homologous sequences from other cell sources.

In order to produce the polypeptides of the invention, the above-definednucleic acid sequences are generally placed under the control of signalswhich enable them to be expressed in a host cell. The choice of thesesignals (promoters, terminators, secretory leader sequence, etc.) mayvary depending on the host cell employed. Preferably, these nucleotidesequences of the invention form part of a vector which can beautonomously replicating or one which integrates. More especially,autonomously replicating vectors can be prepared using sequences whichreplicate autonomously in the chosen host. Integrating vectors can beprepared, for example, using sequences which are homologous to certainregions of the host genome and which enable the vector to integrate bymeans of homologous recombination.

The host cells which can be used for producing the polypeptides of theinvention can be either eucaryotic or procaryotic hosts. Suitableeucaryotic hosts which may be mentioned are animal cells, yeasts orfungi. In particular, yeasts which may be cited are the yeasts of thegenera Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces orHansenula. Animal cells which may be cited are COS, CHO, C127, etc.cells. Fungi which may more particularly be cited are Aspergillus spp.or Trichoderma spp. The following bacteria are preferably used asprocaryotic hosts: E. coli, Bacillus or Streptomyces.

The nucleic acid sequences according to the invention can also beemployed to construct antisense nucleic acids which can be used aspharmaceutical agents. Inhibition of the expression of certain oncogenesby antisense nucleic acids has proved to be a useful strategy inunderstanding the role of these oncogenes and a particularly promisingapproach to achieving an anti-cancer treatment. Antisenseoligonucleotides are small oligonucleotides which are complementary tothe coding strand of a given gene and, for this reason, are able tohybridize specifically with the transcribed mRNA, inhibiting itstranslation into protein. Such oligonucleotides can be constituted byall or part of the nucleic acid sequences defined above. In general,they are sequences or fragments of sequences which are complementary tosequences encoding the peptides according to the invention. Sucholigonucleotides can be obtained from the abovementioned sequences, byfragmentation, etc., or by chemical synthesis.

The invention also relates to the nucleotide probes, as nucleotidesequences, whether or not they are synthetic, which are able tohybridize with the nucleotide sequences defined above which encode apolypeptide of the invention, or with the corresponding mRNA. Suchprobes can be used in vitro as a diagnostic tool. Such probes have to belabelled in advance, and different techniques for doing this are knownto the person skilled in the art. The hybridization conditions underwhich these probes can be used are the normal stringency conditions.These probes can also be used to detect and isolate homologous nucleicacid sequences which encode a polypeptide of the invention, using othercellular sources. As an illustration of these probes, mention may bemade, more particularly, of the probes represented by SEQ ID No. 7 andSEQ ID No. 8, which are used in Example 3 below.

The invention furthermore relates to any pharmaceutical compositionwhich comprises, as its active principle, at least one polypeptide asdefined above.

The invention also relates to any pharmaceutical composition whichcomprises, as its active principle, at least one antibody and/orantibody fragment as defined above, as well as to any pharmaceuticalcomposition which comprises, as its active principle, at least oneantisense oligonucleotide as defined above.

Furthermore, it also relates to the pharmaceutical compositions in whichthe above-defined polypeptides, antibodies and oligonucleotides areattached to each other or to other active principles.

The pharmaceutical compositions according to the invention can be usedto modulate activation of the p21 proteins and, as a result, to modulateproliferation of certain cell types. More especially, thesepharmaceutical compositions are intended for treating cancers. Thus,numerous cancers have been associated with the presence of oncogenic Rasproteins. Of those cancers which most frequently contain mutated Rasgenes, mention may be made, in particular, of adenocarcinomas of thepancreas, 90% of which contain a Ki-Ras oncogene whose twelfth codon ismutated (Almoguera et al., Cell 53 (1988) 549), adenocarcinomas of thecolon and cancers of the thyroid (50%), or carcinomas of the lung andmyeloid leukaemias (30%, Bos, J. L. Cancer Res. 49 (1989) 4682).

The invention also relates to the use of the above-described moleculesfor modulating, that is inhibiting, the activity of the p21 proteins. Inparticular, the invention relates to the use of all, or a fragment, ofG3BP for interfering with the signals which are transduced by theproducts of the Ras genes. The protein fragments which are homologouswith the hnRNPs are advantageously used to inhibit the binding of G3BPto its target RNAs. Sequences which are identical, or complementary, tothese target RNAs can also be used for interfering with the signalswhich are transduced by the G3BP protein.

The invention also provides a process for detecting expression and/oroverexpression of the G3BP protein in a biological sample. Such aprocess comprises, for example, bringing such a sample into contact withan antibody or antibody fragment according to the invention, detectingthe antigen/antibody complexes, and comparing the results which areobtained with a standard sample. In such a process, the antibody can bein suspension or immobilized in advance on a support. This process canalso comprise bringing the sample into contact with a nucleotide probeaccording to the invention, detecting the hybrids which are obtained,and comparing with those obtained in the case of a standard sample.

The present invention can be used in many ways in the therapeuticsphere: since the polypeptides, antibodies and nucleotide sequences ofthe invention are able to modulate the activity of the Ras genes, theythereby make it possible to intervene in the process of cancerdevelopment. Due to the fact that they are strongly expressed instriated skeletal muscle cells, these peptides probably also intervenein pathologies which are linked to a signalling defect such as diabetes,for example. Another aspect of the invention consists in using DNA orRNA nucleotide sequences which are able to interact with the claimedpolypeptides. These sequences can be prepared using the method describedin International Application WO 91/19813.

The invention can also be used in connection with the diagnosis andtyping of cancers.

Other advantages of the present invention will be apparent from readingthe following examples and figures, which are to be considered as beingby way of illustration and not limiting.

FIGURES

FIG. 1: Effect of G3BP overexpression in NIH 3T3 fibroblasts on CATactivity.

FIG. 2: Effect of G3BP overexpression in NIH 3T3 fibroblasts on theformation of foci which is induced by the Src and Ras oncogenes.

MATERIAL AND METHODS General Cloning Techniques

The conventional methods which are used in molecular biology, such aspreparative extractions of plasmid DNA, centrifugation of plasmid DNA ina caesium chloride gradient, electrophoresis on agarose or acrylamidegels, purification of DNA fragments by electroelution, extraction ofproteins with phenol or phenol/chloroform, precipitation of DNA in asaline medium with ethanol or isopropanol, transformation intoEscherichia coli, etc., are well known to the person skilled in the artand are amply described in the literature Maniatis T. et al., "MolecularCloning, a Laboratory Manual", Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F. M. et al., (eds), "CurrentProtocols in Molecular Biology", John Wiley & Sons, New York, 1987!.

The restriction enzymes were supplied by New England Biolabs (Biolabs),Bethesda Research Laboratories (BRL) or Amersham and are used inaccordance with the suppliers' recommendations.

Plasmid pGEX 2T is obtained commercially.

Enzymic amplification of DNA fragments by the technique termed PCRpolymerase-catalysed chain reaction, Saiki R. K. et al., Science 230(1985) 1350-1354; Mullis K. B. et Faloona F. A., Meth. Enzym. 155 (1987)335-350! is performed using a DNA thermal cycler (Perkin Elmer Cetus) inaccordance with the manufacturer's specifications.

The nucleotide sequences are verified by the method developed by Sangeret al. Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467! using the kitdistributed by Amersham.

In general, the normal stringency conditions for the hybridizationexperiments are the following: hybridization: 3×SCC in the presence of5×Denhart's at 65° C.; washing: 0.5×SSC at 65° C.

EXAMPLE 1 Preparation of glutathione-S-transferase SH3 Fusion Proteins

The DNA sequences encoding the SH3 domains of the GAP (residues 275 to350) and c-Src (residues 84 to 148) proteins are amplified by the P.C.R.technique and cloned into an expression vector, pGEX2T, between theBamHI and EcoRI restriction sites. The bacteria which have thus beentransformed are cultured, induced with IPTG(1-thio-β-D-galactopyranoside) and lysed by sonication. The GST SH3fusion proteins are purified by affinity chromatography on glutathioneagarose beads (Pharmacia LKB biotech) and then eluted with 10 mM reducedglutathione.

EXAMPLE 2

1) Preparation of the Cell Lysates

ER22 cells (hamster fibroblasts which overexpress human EGF receptor) orNIH 3T3 cells which express C-Src pp60 (F-527) are cultured on DMEMmedium (Dubelcco's modified Eagle medium), which is enriched with 10%foetal calf serum containing the antibiotic G418 (200 μg/ml) and 2 mM/lglutamine (5GIBCO-BRL), at 370° C. under 5% CO₂.

In each assay, the ER22 cells are cultured in 100 mm dishes until theybecome confluent and are then incubated without serum for 18 hours.Sodium orthovanadate is added to a final concentration of 100 μM and theincubation is continued for 30 minutes. EGF is then added directly tothe medium to give a final concentration of 80 nM over a period of 10minutes and at 37° C.

For certain assays, the mitotic cells are recovered by treating with 0.4μg of nocodazole (SIGMA) per ml for 18 hours. They are rapidly washedwith a phosphate-based saline buffer, recooled in ice and thensolubilized at 4° C. over a period of 30 minutes in 1 ml of lysisbuffer, HNTG (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton×100, 10%glycerol, 1 mM MgCl₂, 1 mM EGTA, in the presence of phosphataseinhibitors (1 mM Na₃ VO₄, 10 mM Na₄ P₂ O₇ and 10 mM NaF) and proteaseinhibitors (1 μg/ml leupeptin, 1 μg/ml trypsin inhibitor, 1 μg/mlpepstatin A, 2 μg/ml aprotinine, 10 μg/ml benzamidine, 1 mMphenylmethanesulfonyl fluoride, 1 μg/ml 1 μg/ml antipain and 1 μg/mlchymostatin).

The lysates are clarified by centrifuging at 15,000 rpm for 10 min. Theprotein concentration is then determined (Biorad microtest).

2) Test for Direct Bonds

The whole of the cell lysate (200 μg) and the immunoprecipitatedproteins are separated on a 7.5% sodium dodecyl sulphate polyacrylamidegel (SDS-PAGE) and then transferred on to a polyvinylidene difluoridemembrane (PVDF, Millipore Corp.). Non-specific binding to the filters isblocked, at room temperature for 2 hours, with 2% skimmed milk in PBScontaining 0.05% Tween 20. The filters are incubated with GST proteinand the GST-SH3 proteins in blocking buffer at 4° C. for 12 h. Afterhaving been washed with PBS-0.05% Tween 20, the bound proteins aredetected by incubating successively with an anti-GST monoclonal antibody(0.25 μg/ml) (Hybridolab Pasteur Inst.), an anti-mouse antibodyconjugated to alkaline phosphatase and 5-bromo-4-chloro-3-indolylphosphate toluidinium nitroblue tetrazolium salt (PROMEGA).

3) Competition Test

Synthetic peptides which correspond to sequences (275-305), (299-326),(317-326) and (320-350) of GAP SH3 or to the putative sequence ofdynamine (RRAPAVPPARPGS), which binds to the SH3 domaine, aresynthesized on an Applied Biosystems 431 A apparatus using FMOCchemistry. The purified p68 protein is isolated by electrophoresis on asodium dodecyl sulphate polyacrylamide gel and transferred byelectrophoresis on to a PVDF membrane. The membranes are incubated withincreasing quantities of peptides. The peptide poly-L-proline (SIGMA) isused at 500 μM in a control reaction. After incubating for 1 h, theprotein GST-GAP-SH3 (2 μg/ml) is added to each reaction medium. Thefiltrates are probed with an anti-GST monoclonal antibody as previouslydescribed.

4) Immunoprecipitation and Immunotransfer

The soluble cell lysates (3 mg) are clarified with 50 μl of protein ASepharose CL-4B (Pharmacia Biotech) at 4° C. for 2 h. The clarified celllysates are incubated with anti-phosphotyrosine monoclonal antibody(monoclonal antibody 4G10--Upstate Biotechnology Incorporated) at 4° C.for 4 h. 50 μl of protein A Sepharose are then added to the complex andthe incubation is continued at 4° C. overnight. The immunoprecipitatematerial is washed 3 times with an HNTG buffer and solubilized in asample of SDS buffer (100 μl). The complexes are then separated bySDS-PAGE and transferred by electrophoresis to PVDF membranes.

The membranes are incubated with phosphotyrosine monoclonal antibody inTBS (10 mM Tris, pH 7.4, 150 nM NaCl, 3% bovine serum albumin) and alsoincubated with second antibodies which are conjugated to alkalinephosphatase. Substrates for the alkaline phosphatase are then added forappropriate colour development.

5) Purification and Analysis of the Sequence Approximately 5.10⁹ ER22cells are lysed in 200 ml of HNTG buffer. The lysate is centrifuged at15,000 g for 15 min and diluted 5 times in an HNG buffer (50 mM Hepes,pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EGTA, phosphatase inhibitors andprotease inhibitors). The lysate is incubated overnight with 6 ml ofFast Flow S-Sepharose which is equilibrated in the same buffer. Thecomplex is transferred to a column (IBF--2.5×1.3 cm). The column iswashed with 10 times its volume of buffer and the bound proteins arethen eluted, at an elution rate of 60 ml/h, with a linear gradient of 60ml of from 0 to 1 M NaCl in the same buffer. The fractions possessingbinding activity, as determined under the conditions of Example 2.2(0.15-0.37 M NaCl), are collected, diluted 10 times in an HNG buffer andloaded on to Heparin-Sepharose Cl-6B (3 ml) (Pharmacia LKB), which ispreequilibrated with the same buffer at an elution rate of 24 ml/h.After having been washed with the HNG buffer, the column is eluted witha 24 ml gradient of from 0 to 1 M NaCl. Active fractions from the S FastFlow chromatography are collected, diluted 4 times in an MES buffer (100mM MES, pH 6.8, 1 mM MgSO₄, 1 mM EGTA) and transferred to an agarose ATPcolumn (3 ml) (Sigma No. A9264) which has been preequilibrated with theMES buffer containing 50 mM NaCl at an elution rate of 6 ml/h. Thecolumn is then washed with 20 ml of MES buffer containing 50 mM NaCl andeluted at 30 ml/h with a linear gradient of from 50 mM to 2M NaCl in theMES buffer. The p68 protein elutes between 0.3M and 0.4M NaCl. Theactive fractions are collected, dialysed with 20 mM NH₄ HCO₃, pH 8.3,and concentrated. After having been taken to dryness, the proteins areresuspended in an SDS buffer and separated by electrophoresis on apolyacrylamide gel. The gel is stained with Coomassie blue and the 68kDA molecular weight band, corresponding to the SH₃ binding activity, isrecovered. It is washed for 1 hour with the following solutions: water,water/methanol (90/10), water/CH₃ CN (80/20) and water/CH₃ CN (50/50).

The gel band, which contains the purified p68 protein, is then dividedinto small fragments and dried under a SPEED/VAC (SAVANT). 400 μl of asolution containing 25 mM Tris, pH 8.5, 1 mM EDTA, 0.05% SDS and 5 μg ofLys-c endoproteinase (Boehringer Mannheim) are added, and the whole isincubated overnight at 37° C. The hydrolysate is injected on to areverse phase HPLC column (Vydac C18: 2.1×250 mm). The column is elutedin 150 minutes, at 0.2 ml/min, with a linear gradient of from 0 to 35% B(A:H₂₀ +0.07% TFA, and B: CH₃ CN+0.07% TFA) and the elution peaksobserved at retention times of 113.7, 117.7 and 133.7 min are collectedand sequenced directly using an Applied Biosystems 477A microsequencer.The corresponding peptide sequence obtained in this way is presented inSEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. Thesesequences do not exhibit any homology with proteins referenced in theprotein databases (PIR 33. Swiss-Prot 33 (intelligenetics)).

In order to determine whether the p68 protein is tyrosine-phosphorylatedin the mitotic or non-synchronous cells, the proteins in the lysatesderived from ER22 cells which are cultured in 10% foetal calf serum orfrom cells treated with nocodazole are immunoprecipitated withanti-phosphotyrosine antibodies, transferred to membranes and eitherimmunodetected with anti-phosphotyrosine antibodies or incubated witheither GST-GAP-SH3 or GST-Src-SH3 under the conditions described inExamples 2.2 and 2.4. As a control, the proteins of NIH 3T3 cells whichhave been transformed with an activated allele of c-Src (c-Src Y527F)are tested under the same conditions as those described for the ER22proteins. The results which are obtained demonstrate that proteins aretyrosine-phosphorylated, more especially in the 60-70 kDa region. Nobinding to a phosphorylated p68 protein is detected in the ER22 cellsusing the GST-GAP-SH3 or GST-Src-SH3 probes. In the NIH3T3 cells(c-Src-Y527F), the GST-Src-SH3 probe binds to a tyrosine-phosphorylatedprotein of 68 kDa molecular weight whereas the GST-GAP-SH3 probe doesnot interact with any protein.

Competition experiments to confirm the functional involvement of thisprotein in the Ras signalling pathway were carried out under theconditions described in Example 2.3. The results which are obtaineddemonstrate that the peptides derived from GAP are able to blockRas-induced rupture of Xenopus egg germinal vesicles (GVBD); GAP SH3peptides (299-326) and (317-326) are also able to block the interactionbetween the G3BP protein and GAP.

These results clearly confirm that the ability to block, or interferewith, the activity of the protein according to the invention constitutesa particularly promising, novel approach in the treatment of cancer.

EXAMPLE 3 Isolation of the Human SEQ ID No. 6 Sequence from a HumanPlacenta cDNA Library.

Sequences SEQ ID No. 7 and No. 8, which were based on peptide sequencesSEQ ID No. 1 and SEQ ID No. 3, are used as probes. The DNA of theClonetech HL1008B library was prepared and used in a PCR reaction. 30amplification cycles were carried out under the following conditions:denaturation, 1 min at 94° C., annealing, 1 min between 35 and 40° C.,and elongation, 1 min at 72° C. This reaction gives rise to a 1.3 kb DNAfragment one part of whose sequence is given by SEQ ID No. 6. A NorthernBlot analysis demonstrates that the corresponding RNA (3.3 kb) isubiquitous and is expressed very strongly in adult skeletal muscle.

EXAMPLE 4 Isolation of the SEQ. ID No. 10 Sequence, which Encodes Humanp68, from a Human Placenta cDNA Library.

The two oligonucleotides (SEQ ID No. 7) and (SEQ ID No. 8) are used asprimers for amplifying a cDNA in a human placenta library. The PCR wascarried out using Perkin Elmer Amplitaq at an annealing temperature of35° C. followed by a 1 min extension at 72° C. in the presence of 10%formamide. We amplified a cDNA fragment of 1164 bp. After cloningdirectly into a pMOSblue vector (Amersham), which contained forward andreverse -20 sequences, the fragment was sequenced using fluorescentprobes. This PCR fragment was then employed as a probe for screening 10⁶phages from a human placenta λgt11 cDNA library (Clonetech). The probewas synthesized by the Amersham Rediprime system and the filters wereincubated at 45° C. for 16 hours in hybridization buffer (6×SSC,5×Denhardt's, 100 μg/ml salmon sperm, 0.25% SDS) containing the probe.The filters were then washed at room temperature for 1 hour and athybridization temperature for 20 min in 2×SSC, 0.05% SDS. Eight positiveclones were identified. Two of them were purified and their cDNA wasdigested with EcoRI in order to excise the inserts. They were subclonedinto M13mpl8/EcoRI in order to be sequenced. The sequencing was carriedout on fragments obtained by progressive deletion of the cDNA withexonuclease III (Nested Deletion Kit, Pharmacia Biotech). The sizedifferences of the fragments were analysed by PCR amplification betweenthe forward and reverse -20 primers of the M13 vector, and differentsize populations were selected for sequencing. By assembling thedifferent sequences which were obtained, it was possible to reconstitutethe entire open reading frame of p68.

EXAMPLE 5 Overexpression of G3BP in NIH 3T3 Fibroblasts

NIH 3T3 fibroblasts are transfected with a reporter gene, that ofchloramphenicol acetyl transferase, which is placed under the control ofRas response elements which derive from the polyome virus enhancer.These elements are stimulated from 15 to 30 fold when the cells aretransfected with an expression vector carrying the cDNA of the Src andRas oncogenes. These stimulations are modified when the G3BP protein isexpressed following cotransfection with an expression vector whichcontains a cDNA which corresponds to the open reading frame of theprotein. The G3BP inhibits, in a dose-dependent manner, the CAT activitywhich is stimulated by Src and by the oncogenic form of Ras. Thisobservation is presented in FIG. 1.

In the same way, expression of the G3BP protein opposes the formation offoci which is induced by the Src and Ras oncogenes. FIG. 2 gives anaccount of this observation.

These experiments clearly demonstrate the ability of G3BP to oppose theproliferative effects of the signals which are transduced by the normalor oncogenic Ras proteins.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 10    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ValMetGluLysProSerProLeuLeuValGlyArgGluPheValArg    151015    GlnTyrIle    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    XaaXaaGluGlyAspAspArgAspAsnArgLeuLeuGlyPro    1510    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    LeuProAsnPheGlyPheValValPheAspAspSerGluProValGln    151015    Lys    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    SerAlaThrProAlaProAlaAspValAlaProAlaGlnGluAspLeu    151015    ArgXaaPhe    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 68 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS:    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    ArgGluAlaGlyGluGlnGlyAspIleGluProArgArgMetValArg    151015    HisProAspSerHisGlnLeuPheIleGlyAsnLeuProHisGluVal    202530    AspLysSerGluLeuLysAspPhePheGlnSerTyrGlyAsnValVal    354045    GluLeuArgIleAsnSerGlyProLysLeuProAsnPheAlaPheVal    505560    ValPheAspAsp    65    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 204 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Oligonucleotide"    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..204    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CGTGAGGCTGGTGAGCAAGGTGACATTGAACCCCGAAGAATGGTGAGACACCCTGACAGT60    CACCAACTCTTCATTGGCAACCTGCCTCATGAAGTGGACAAATCAGAGCTTAAAGATTTC120    TTTCAAAGTTATGGAAACGTGGTGGAGTTGCGCATTAACAGTGGTGGGAAATTACCCAAT180    TTCGCCTTCGTCGTCTTCGATGAT204    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Oligonucleotide"    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 1..32    (D) OTHER INFORMATION: /mod.sub.-- base=i    /note= "N=Inosine"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GTNATGGANAANCCNTCCCCNCTNCTNGTNGG32    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "Oligonucleotide"    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..23    (ix) FEATURE:    (A) NAME/KEY: modified.sub.-- base    (B) LOCATION: 1    (D) OTHER INFORMATION: /mod.sub.-- base=i    /note= "N=Inosine"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GAATCATCGAANACNACGAANCCGAA26    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 466 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    MetValMetGluLysProSerProLeuLeuValGlyArgGluPheVal    151015    ArgGlnTyrTyrThrLeuLeuAsnGlnAlaProAspMetLeuHisArg    202530    PheTyrGlyLysAsnSerSerTyrValHisGlyGlyLeuAspSerAsn    354045    GlyLysProAlaAspAlaValTyrGlyGlnLysGluIleHisArgLys    505560    ValMetSerGlnAsnPheThrAsnCysHisThrLysIleArgHisVal    65707580    AspAlaHisAlaThrLeuAsnAspGlyValValValGlnValMetGly    859095    LeuLeuSerAsnAsnAsnGlnAlaLeuArgArgPheMetGlnThrPhe    100105110    ValLeuAlaProGluGlySerValAlaAsnLysPheTyrValHisAsn    115120125    AspIleArgTyrGlnAspGluValPheGlyGlyPheValThrGluPro    130135140    GlnGluGluSerGluGluGluValGluGluProGluGluArgGlnGln    145150155160    ThrProGluValValProAspAspSerGlyThrPheTyrAspGlnAla    165170175    ValValSerAsnAspMetGluGluHisLeuGluGluProValAlaGlu    180185190    ProGluProAspProGluProGluProGluGlnGluProValSerGlu    195200205    IleGlnGluGluLysProGluProValLeuGluGluThrAlaProGlu    210215220    AspAlaGlnLysSerSerSerProAlaProAlaAspIleAlaGlnThr    225230235240    ValGlnGluAspLeuArgThrPheSerTrpAlaSerValThrSerLys    245250255    AsnLeuProProSerGlyAlaValProValThrGlyIleProProHis    260265270    ValValLysValProAlaSerGlnProArgProGluSerLysProGlu    275280285    SerGlnIleProProGlnArgProGlnArgAspGlnArgValArgGlu    290295300    GlnArgIleAsnIleProProGlnArgGlyProArgProIleArgGlu    305310315320    AlaGlyGluGlnGlyAspIleGluProArgArgMetValArgHisPro    325330335    AspSerHisGlnLeuPheIleGlyAsnLeuProHisGluValAspLys    340345350    SerGluLeuLysAspPhePheGlnSerTyrGlyAsnValValGluLeu    355360365    ArgIleAsnSerGlyGlyLysLeuProAsnPheGlyPheValValPhe    370375380    AspAspSerGluProValGlnLysValLeuSerAsnArgProIleMet    385390395400    PheArgGlyGluValArgLeuAsnValGluGluLysLysThrArgAla    405410415    AlaArgGluGlyAspArgArgAspAsnArgLeuArgGlyProGlyGly    420425430    ProArgGlyGlyLeuGlyGlyGlyMetArgGlyProProArgGlyGly    435440445    MetValGlnLysProGlyPheGlyValGlyArgGlyLeuAlaProArg    450455460    GlnGlx    465    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2129 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..2129    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GCTTGCCTGTCAGGTCGACTCTAGAGCCCGGGTACCGAGCTCGAATTCGGCGGGGTTTGT60    ACTATCCTCGGTGCTGTGGTGCAGAGCTAGTTCCTCTCCAGCTCAGCCGCGTAGGTTTGG120    ACATATTTACTCTTTTCCCCCCAGGTTGAATTGACCAAAGCAATGGTGATGGAGAAGCCT180    AGTCCCCTGCTGGTCGGGCGGGAATTTGTGAGACAGTATTACACACTGCTGAACCAGGCC240    CCAGACATGCTGCATAGATTTTATGGAAAGAACTCTTCTTATGTCCATGGGGGATTGGAT300    TCAAATGGAAAGCCAGCAGATGCAGTCTACGGACAGAAAGAAATCCACAGGAAAGTGATG360    TCACAAAACTTCACCAACTGCCACACCAAGATTCGCCATGTTGATGCTCATGCCACGCTA420    AATGATGGTGTGGTAGTCCAGGTGATGGGGCTTCTCTCTAACAACAACCAGGCTTTGAGG480    AGATTCATGCAAACGTTTGTCCTTGCTCCTGAGGGGTCTGTTGCAAATAAATTCTATGTT540    CACAATGATATCTTCAGATACCAAGATGAGGTCTTTGGTGGGTTTGTCACTGAGCCTCAG600    GAGGAGTCTGAAGAAGAAGTAGAGGAACCTGAAGAAAGCAGCAAACACCTGAGGTGGTAC660    CTGATGATTCTGGAACTTTCTATGATCAGGCAGTTGTCAGTAATGACATGGAAGAACATT720    TAGAGGAGCCTGTTGCTGAACCAGAGCCTGATCCTGAACCAGAACCAGAACAAGAACCTG780    TATCTGAAATCCAAGAGGAAAAGCCTGAGCCAGTATTAGAAGAAACTGCCCCTGAGGATG840    CTCAGAAGAGTTCTTCTCCAGCACCTGCAGACATAGCTCAGACAGTACAGGAAGACTTGA900    GGACATTTTCTTGGGCATCTGTGACCAGTAAGAATCTTCCACCCAGTGGAGCTGTTCCAG960    TTACTGGGATACCACCTCATGTTGTTAAAGTACCAGCTTCACAGCCCCGTCCAGAGTCTA1020    AGCCTGAATCTCAGATTCCACCACAAAGACCTCAGCGGGATCAAAGAGTGCGAGAACAAC1080    GAATAAATATTCCTCCCCAAAGGGGACCCAGACCAATCCGTGAGGCTGGTGAGCAAGGTG1140    ACATTGAACCCCGAAGAATGGTGAGACACCCTGACAGTCACCAACTCTTCATTGGCAACC1200    TGCCTCATGAAGTGGACAAATCAGAGCTTAAAGATTTCTTTCAAAGTTATGGAAACGTGG1260    TGGAGTTGCGCATTAACAGTGGTGGGAAATTACCCAATTTTGGTTTTGTTGTGTTTGATG1320    ATTCTGAGCCTGTTCAGAAAGTCCTTAGCAACAGGCCCATCATGTTCAGAGGTGAGGTCC1380    GTCTGAATGTCGAAGAGAAGAAGACTCGAGCTGCCAGGGAAGGCGACCGACGAGATAATC1440    GCCTTCGGGGACCTGGAGGCCCTCGAGGTGGGCTGGGTGGTGGAATGAGAGGCCCTCCCC1500    GTGGAGGCATGGTGCAGAAACCAGGATTTGGAGTGGGAAGGGGGCTTGCGCCACGGCAGT1560    AATCTTCATGGATCTTCATGCAGCCATACAAACCCTGGTTCCAACAGAATGGTGAATTTT1620    CGACAGCCTTTGGTATCTTGGAGTATGACCCCAGTCTGTTATAAACTGCTTAAGTTTGTA1680    TAATTTTACTTTTTTTGTGTGTTAATGGTGTGTGCTCCCTCTCCCTCTCTTCCCTTTCCT1740    GACCTTTAGTCTTTCACTTCCAATTTTGTGGAATGATATTTTAGGAATAACGGACTTTTA1800    CCCGAATTCGTAATCATGGTCATAGCTGTTTCCGTGTGAAATTGTTATCCGCTCACAATT1860    CCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGC1920    TAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGC1980    CAGCGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGG2040    GTGGTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCTGGCCCTG2100    AGAGAGTTGCAGCAAGCGGTCCACGCTGG2129    __________________________________________________________________________

We claim:
 1. An isolated polypeptide which binds an SH3 domain of GAPprotein, wherein the polypeptide comprises:a) a peptide sequenceselected from among the sequences SEQ ID No. 2, SEQ ID No. 3, SEQ No. 4,SEQ ID No. 5 and SEQ ID No. 9, or b) SEQ ID No.
 1. 2. The polypeptideaccording to claim 1 which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3 or SEQ ID No. 4, or a combination thereof.
 3. The polypeptideaccording to claim 1, characterized in that it has a molecular weight ofthe order of 68 kDa.
 4. The polypeptide according to claim 1 of humanorigin.
 5. The polypeptide according to claim 4 which comprises thesequence SEQ ID No. 5 or SEQ ID No.
 9. 6. The polypeptide according toclaim 1 having a sequence of SEQ. ID No.
 9. 7. The polypeptide accordingto claim 1 having one or more tyrosine motifs, which are notphosphorylated in mitotic or growing cells.